The long-range goal of this project is to define the interactions in the transcription complex that allow regulation of pausing and termination by human RNA polymerase II. Pausing and termination are the targets of specialized regulatory proteins that modify the transcription complex to allow synthesis of complete mRNA chains. The mechanisms by which these regulatory proteins alter the activity of RNA polymerase II are unknown. To provide a conceptual framework within which these mechanisms can be defined, we are elucidating the fundamental mechanism of a model pause signal from the early transcribed region of HIV-1. Defining the HIV-1 pause mechanism in structural detail will then allow us to determine how regulatory proteins affect the response of RNA polymerase II to the pause signal. However, a structure/function study of pause mechanisms requires the ability to obtain human RNA polymerase II enzymes with specifically altered amino-acids sequences. The overall goal of the project is to make several human RNA polymerase II mutants and use them to test models of transcriptional pausing and the mechanisms of regulatory protein action. We now have established a method by which we can produce recombinant mutant human RNA polymerase II, and have on hand a mutant enzyme that is defective for elongation. This and other proposed mutant enzymes will be useful to study not only the basic mechanism of pausing, but also the interactions of protein factors that regulate pausing. The specific aims of the project are (i) use altered human RNA polymerase II enzymes to define mechanism of pausing and regulatory protein action;(ii) dissect the mechanism of action of a negative elongation factor;and (iii) use tagged RNA polymerase II for single molecule analysis of transcription elongation by human RNA polymerase II. It is well established that unregulated transcription elongation by human RNA polymerase II is a major factor in deadly human diseases like AIDS and certain cancers. However, little is known about how the elongation properties of RNA polymerase II are altered in such diseases. Our research is aimed at understanding these fundamental mechanisms of transcription elongation. The knowledge gained will increase understanding of the basic molecular biology of human diseases.